The present invention relates a method of reducing levels of TNFxcex1 in a mammal and to compounds and compositions useful therein.
TNFxcex1, or tumor necrosis factor xcex1, is a cytokine which is released primarily by mononuclear phagocytes in response to various immunostimulators. When administered to animals or humans it causes inflammation, fever, cardiovascular effects, hemorrhage, coagulation and acute phase responses similar to those seen during acute infections and shock states.
Excessive or unregulated TNFxcex1 production has been implicated in a number of disease conditions. These include endotoxemia and/or toxic shock syndrome {Tracey et al., Nature 330, 662-664 (1987) and Hinshaw et al., Circ. Shock 30, 279-292 (1990)}; cachexia {Dezube et al., Lancet, 335(8690), 662 (1990)}; and Adult Respiratory Distress Syndrome where TNFxcex1 concentration in excess of 12,000 pg/milliliters have been detected in pulmonary aspirates from ARDS patients {Millar et al., Lancet 2(8665), 712-714 (1989)}. Systemic infusion of recombinant TNFxcex1 also resulted in changes typically seen in ARDS {Ferrai-Baliviera et al., Arch Surg. 124(12), 1400-1405 (1989)}.
TNFxcex1 appears to be involved in bone resorption diseases, including arthritis where it has been determined that when activated, leukocytes will produce a bone-resorbing activity, and data suggest that TNFxcex1 contributes to this activity. {Bertolini et al, Nature 319, 516-518 (1986) and Johnson et al, Endocrinology 124(3), 1424-1427 (1989).} It has been determined that TNFxcex1 stimulates bone resorption and inhibits bone formation in vitro and in vivo through stimulation of osteoclast formation and activation combined with inhibition of osteoblast function. Although TNFxcex1 may be involved in many bone resorption diseases, including arthritis, the most compelling link with disease is the association between production of TNFxcex1 by tumor or host tissues and malignancy associated hypercalcemia {Calci. Tissue Int. (US) 46(Suppl.), S3-10 (1990)}. In Graft versus Host Reaction, increased serum TNFxcex1 levels have been Rated with major complication following acute allogenic bone marrow transplants {Holler et at, Blood, 75(4), 1011-1016 (1990)}.
Cerebral malaria is a lethal hyperacute neurological syndrome associated with high blood levels of TNFxcex1 and the most severe complication occurring in malaria patients. Levels of serum TNFxcex1 correlated directly with the severity of disease and the prognosis in patients with acute malaria attacks {Grau et al., N. Engl. J. Med, 320(24), 1586-1591 (1989)}.
TNFxcex1 also plays a role in the area of chronic pulmonary inflammatory diseases. The deposition of silica particles leads to silicosis, a disease of progressive respiratory failure caused by a fibrotic reaction. Antibody to TNFxcex1 completely blocked the silica-induced lung fibrosis in mice {Pignet et al., Nature, 344:245-247 (1990)}. High levels of TNFxcex1 production (in the serum and in isolated macrophages) have been demonstrated in animal models of silica and asbestos induced fibrosis {Bissonnette et al., Inflammation 13(3), 329-339 (1989)}. Alveolar macrophages from pulmonary sarcoidosis patients have also been found to spontaneously release massive quantities of TNFxcex1 as compared with macrophages from normal donors {Baughman et al., J. Lab. Clim Med. 115(1), 36-42 (1990)}.
TNFxcex1 is also implicated in the inflammatory response which follows reperfusion, called reperfusion injury, and is a major cause of tissue damage after loss of blood flow {Vedder et al., PNAS 87, 2643-2646 (1990)}. TNFxcex1 also alters the properties of endothelial cells and has various pro-coagulant activities, such as producing an increase in tissue factor pro-coagulant activity and suppression of the anticoagulant protein C pathway as well as down-regulating the expression of thrombomodulin {Sherry et at, J. Cell Biol. 107, 1269-1277 (1988)}. TNFxcex1 has pro-inflammatory activities which together with its early production (during the initial stage of an inflammatory event) make it a likely mediator of tissue injury in several important disorders including but not limited to, myocardial infarction, stroke and circulatory shock. Of specific importance may be TNFxcex1-induced expression of adhesion molecules, such as intercellular adhesion molecule (ICAM) or endothelial leukocyte adhesion molecule (ELAM) on endothelial cells {Munro et al., Am. J. Path. 135(1), 121-132 (1989)}. Moreover, it now is known that TNFxcex1 is a potent activator of retrovirus replication including activation of HIV-1. {Duh et al., Proc. Nat. Acad. Sci. 86, 5974-5978 (1989); Poll et al., Proc. Nat. Acad. Sci. 87, 782-785 (1990); Monto et al., Blood 79, 2670 (1990); Clouse et al., J. Immunol. 142, 431-438 (1989); Poll et al., AIDS Res. Hum. Retrovirus, 191-197 (1992)}. AIDS results from the infection of T lymphocytes with Human Immunodeficiency Virus (HIV). At least three types or strains of HIV have been identified, Le., HIV-1, HIV-2 and HIV-3. As a consequence of HIV infection, T-cell mediated immunity is impaired and infected individuals manifest severe opportunistic infections and/or unusual neoplasms. HIV entry into the T lymphocyte requires T lymphocyte activation. Other viruses, such as HIV-1, HIV-2 infect T lymphocytes after T cell activation and such virus protein expression and/or replication is mediated or maintained by such T cell activation. Once an activated T lymphocyte is infected with HIV, the T lymphocyte must continue to be maintained in an activated state to permit HIV gene expression and/or HIV replication. Cytokines, specifically TNFxcex1, are implicated in activated T-cell mediated HIV protein expression and/or virus replication by playing a role in maintaining T lymphocyte activation. Therefore, interference with cytokine activity such as by prevention or inhibition of cytokine production, notably TNFxcex1, in an HIV-infected individual aids in limiting the maintenance of T lymphocyte caused by HIV infection.
Monocytes, macrophages, and related cells, such as kupffer and glial cells, have also been implicated in maintenance of the HIV infection. These cells, like T cells, are targets for viral replication and the level of viral replication is dependent upon the activation state of the cells. {Rosenberg et al, The Immunopathogenesis of HIV Infection, Advances in Immunology, 57 (1989)}. Cytokines, such as TNFxcex1, have been shown to activate HIV replication in monocytes and/or macrophages {Poli et al. Proc. Natl. Acad. Sci., 87, 782-784 (1990)}, therefore, prevention or inhibition of cytokine production or activity aids in limiting HIV progression as stated above for T cells. Additional studies have identified TNFxcex1 as a common factor in the activation of HIV in vitro and has provided a clear mechanism of action via a nuclear regulatory protein found in the cytoplasm of cells (Osborn, et al., PNAS86, 2336-2340). This evidence suggests that a reduction of TNFxcex1 synthesis may have an antiviral effect in HIV infections by reducing the transcription and thus virus production.
AIDS viral replication of latent HIV in T cell and macrophage lines can be induced by TNFxcex1 {Folks et al., PNAS 86, 2365-2368 (1989)}. A molecular mechanism for the virus inducing activity is suggested by TNFxcex1""s ability to activate a gene regulatory protein (NFxcexaB) found in the cytoplasm of cells, which promotes HIV replication through binding to a viral regulatory gene sequence (LTR) {Osborn et al., PNAS 86, 2336-2340 (1989)}. TNFxcex1 in AIDS and cancer associated cachexia is suggested by elevated serum TNFxcex1 and high levels of spontaneous TNFxcex1 production in peripheral blood monocytes from patients {Wright et al. J. Immunol. 141(1), 99-104 (1988)}. Eur J. Gastroen Hepat 6(9), 821-829, 1994.
TNFxcex1 has been implicated in various roles with other viral infections, such as the cytomegalia virus (CMV), influenza virus, adenovirus, and the herpes family of viruses for similar reasons as those noted.
Preventing or inhibiting the production or action of TNFxcex1 is, therefore, predicted to be a potent therapeutic strategy for many inflammatory, infectious, immunological or malignant diseases. These include but are not restricted to septic shock, sepsis, endotoxic shock, hemodynamic shock and sepsis syndrome, post ischemic reperfusion injury, malaria, mycobacterial infection, meningitis, psoriasis, congestive heart failure, fibrotic disease, cachexia, graft rejection, cancer, autoimmune disease, opportunistic infections in AIDS, rheumatoid arthritis, rheumatoid spondylitis, osteoarthritis, other arthritic conditions, Crohn""s disease, ulcerative colitis, multiple sclerosis, systemic lupus erythrematosis, ENL in leprosy, radiation damage, and hyperoxic alveolar injury. Efforts directed to the suppression of the effects of TNFxcex1 have ranged from the utilization of steroids such as dexamethasone and prednisolone to the use of both polyclonal and monoclonal antibodies {Beutler et al., Science 234, 470-474 (1985); WO 92/11383}. (Clinical and Experimental Rheumatology 1993, 11 (Suppl. 8), 5173-5175). (PNAS 1992, 89, 9784-88). (Annals of the Rheumatic Diseases 1990, 49, 480-486).
The nuclear factor xcexaB (NExcexaB) is a pleiotropic transcriptional activator (Lenardo, et al. Cell 1989, 58, 227-29). NFxcexaB has been implicated as a transcriptional activator in a variety of disease and inflammatory states and is thought to regulate cytokine levels including but not limited to TNFxcex1 and also to be an activator of HIV transcription (Dbaibo, et al. J. Biol. Chem. 1993, 17762-66; Duh et al. Proc. Natl. Acad. Sci. 1989, 86, 5974-78; Bachelerie et al. Nature 1991, 350, 709-12; Boswas et al. J. Acquired Immune Deficiency Syndrome 1993, 6, 778-786; Suzuki et al. Biochem. And Biophys. Res. Comm. 1993, 193, 277-83; Suzuki et al. Biochem. And Biophys. Res Comm. 1992, 189, 1709-15; Suzuki et a., Biochem. Mol. Bio. Int. 1993, 31(4), 693-700; Shakhov et al. 1990, 171, 35-47; and Staal et al Proc. Natl. Acad. Sci. USA 1990, 87, 9943-47). Thus, inhibition of NFxcexaB binding can regulate transcription of cytokine gene(s) and through this modulation and other mechanisms be useful in the inhibition of a multitude of disease states. The compounds claimed in this patent can inhibit the action of NFxcexaB in the nucleus and thus are useful in the treatment of a variety of diseases including but not limited to rheumatoid arthritis, rheumatoid spondylitis, osteoarthritis, other arthritic conditions, septic shock, septis, endotoxic shock, graft versus host disease, wasting, Crohn""s disease, ulcerative colitis, multiple sclerosis, systemic lupus erythrematosis, ENL in leprosy, HIV, AIDS, and opportunistic infections in AIDS.
TNFxcex1 and NFxcexaB levels are influenced by a reciprocal feedback loop. As noted above, the compounds of the present invention affect the levels of both TNFxcex1 and NFxcexaB. It is not known at this time, however, how the compounds of the present invention regulate the levels of TNFxcex1, NFxcexaB, or both.
The present invention is based on the discovery that a class of non-polypeptide imides more fully described herein appear to inhibit the action of TNFxcex1.
The present invention pertains to compounds of the formula: 
in which:
R1 is (i) straight, branched, or cyclic alkyl of 1 to 12 carbon atoms, (ii) phenyl or phenyl substituted with one or more substituents each selected independently of the other from nitro, cyano, trifluoromethyl, carbethoxy, carbomethoxy, carbopropoxy, acetyl, carbamoyl, acetoxy, carboxy, hydroxy, amino, straight or branched alkyl of 1 to 10 carbon atoms, alkoxy of 1 to 10 carbon atoms, or halo, (iii) benzyl or benzyl substituted with one or more substituents each selected independently of the other from nitro, cyano, trifluoromethyl, carbethoxy, carbomethoxy, carbopropoxy, acetyl, carbamoyl, acetoxy, carboxy, hydroxy, amino, alkyl of 1 to 10 carbon atoms, alkoxy of 1 to 10 carbon atoms, or halo, or (iv) xe2x80x94Yxe2x80x94Ph where Y is a straight, branched, or cyclic alkyl of 1 to 12 carbon atoms and Ph is phenyl or phenyl substituted with one or more substituents each selected independently of the other from nitro, cyano, trifluoromethyl, carbethoxy, carbomethoxy, carbopropoxy, acetyl, carbamoyl, acetoxy, carboxy, hydroxy, amino, alkyl of 1 to 10 carbon atoms, alkoxy of 1 to 10 carbon atoms, or halo;
R2 is xe2x80x94H, a branched or unbranched alkyl of 1 to 10 carbon atoms, phenyl, pyridyl, heterocycle, xe2x80x94CH2xe2x80x94Aryl, or xe2x80x94CH2xe2x80x94heterocycle;
R3 is i) ethylene, ii) vinylene, iii) a branched alkylene of 3 to 10 carbon atoms, iv) a branched alkenylene of 3 to 10 carbon atoms, v) cycloalkylene of 4 to 9 carbon atoms unsubstituted or substituted with 1 to 2 substituents each selected independently from nitro, cyano, trifluoromethyl, carbethoxy, carbomethoxy, carbopropoxy, acetyl, carbamoyl, acetoxy, carboxy, hydroxy, amino, substituted amino, alkyl of 1 to 4 carbon atoms, alkoxy of 1 to 4 carbon atoms, or halo, vi) cycloalkenylene of 4 to 9 carbon atoms unsubstituted or substituted with 1 to 2 substituents each selected independently from nitro, cyano, trifluoromethyl, carbethoxy, carbomethoxy, carbopropoxy, acetyl, carbamoyl, acetoxy, carboxy, hydroxy, amino, substituted amino, alkyl of 1 to 4 carbon atoms, alkoxy of 1 to 4 carbon atoms, or halo, or vii) o-phenylene unsubstituted or substituted with 1 to 2 substituents each selected independently from nitro, cyano, trifluoromethyl, carbethoxy, carbomethoxy, carbopropoxy, acetyl, carbamoyl, acetoxy, carboxy, hydroxy, amino, substituted amino, alkyl of 1 to 4 carbon atoms, alkoxy of 1 to 4 carbon atoms, or halo; and,
R4 is xe2x80x94CX, or xe2x80x94CH2xe2x80x94;
X is O or S.
The term alkyl as used herein denotes a univalent saturated branched or straight hydrocarbon chain. Unless otherwise stated, such chains can contain from 1 to 18 carbon atoms. Representative of such alkyl groups are methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, tert-pentyl, hexyl, isohexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, and the like. When qualified by xe2x80x9clowerxe2x80x9d, the alkyl group will contain from 1 to 6 carbon atoms. The same carbon content applies to the parent term xe2x80x9calkanexe2x80x9d and to derivative terms such as xe2x80x9calkoxyxe2x80x9d.
The compounds can be prepared using methods which are known in general for the preparation of imides. General reaction schemes include the reaction of the substituted amine with either phthalic anhydride, N-carbethoxyphthalimide, 1,2-benzenedicarbaldehyde or various substituted anhydrides as illustrated by the formulas: 
R6 and R7 are hydrogen, nitro, cyano, trifluoromethyl, carbethoxy, carbomethoxy, carbopropoxy, acetyl, carbamoyl, acetoxy, carboxy, hydroxy, amino, substituted amino, alkyl of 1 to 4 carbon atoms, alkoxy of 1 to 4 carbon atoms, halo or R6 and R7 together with the carbons to which they are attached represent a cycloalkylene ring of 4 to 9 carbon atoms unsubstituted or substituted with one or more substituents each selected independently from nitro, cyano, trifluoromethyl, carbethoxy, carbomethoxy, carbopropoxy, acetyl, carbamoyl, acetoxy, carboxy, hydroxy, amino, substituted amino, alkyl of 1 to 4 carbon atoms, alkoxy of 1 to 4 carbon atoms, or halo.
A first preferred subclass of Formula I pertains to compounds in which:
R1 is 3,4-diethoxyphenyl and 3,4-dimethoxyphenyl
R3 is o-phenylene substituted with amino; and,
R4 is xe2x80x94COxe2x80x94or xe2x80x94CH2xe2x80x94:
The compounds can be used, under the supervision of qualified professionals, to inhibit the undesirable effects of TNFxcex1. The compounds can be administered orally, rectally, or parenterally, alone or in combination with other therapeutic agents including antibiotics, steroids, etc., to a mammal in need of treatment. Oral dosage forms include tablets, capsules, dragees, and similar shaped, compressed pharmaceutical forms. Isotonic saline solutions containing 20-100 milligrams/milliliter can be used for parenteral administration which includes intramuscular, intrathecal, intravenous and intra-arterial routes of administration. Rectal administration can be effected through the use of suppositories formulated from conventional carriers such as cocoa butter.
Dosage regimens must be titrated to the particular indication, the age, weight, and general physical condition of the patient, and the response desired but generally doses will be from about 1 to about 500 milligrams/day as needed in single or multiple daily administration. In general, an initial treatment regimen can be copied from that known to be effective in interfering with TNFxcex1 activity for other TNFxcex1 mediated disease states by the compounds of the present invention. Treated individuals will be regularly checked for T cell numbers and T4/T8 ratios and/or measures of viremia such as levels of reverse transcriptase or viral proteins, and/or for progression of cytokine-mediated disease associated problems such as cachexia or muscle degeneration. If no effect is found following the normal treatment regimen, then the amount of cytokine activity interfering agent administered is increased, e.g., by fifty percent a week.
The compounds of the present invention also can be used topically in the treatment or prophylaxis of topical disease states mediated or exacerbated by excessive TNFxcex1 production, respectively, such as viral infections, such as those caused by the herpes viruses, or viral conjunctivitis, etc.
The compounds also can be used in the veterinary treatment of mammals other than humans in need of prevention or inhibition of TNFxcex1 production. TNFxcex1 mediated diseases for treatment, therapeutically or prophylactically, in animals include disease states such as those noted above, but in particular viral infections. Examples include feline immuno-deficiency virus, equine infectious anaemia virus, caprine arthritis virus, visna virus, and maedi virus, as well as other lentiviruses.
Certain of these compounds possess centers of chirality and can exist as optical isomers. Both the racemates of these isomers and the individual isomers themselves, as well as diastereomers when there are two chiral centers, are within the scope of the present invention. The racemates can be used as such or can be separated into their individual isomers mechanically as by chromatography using a chiral absorbent. Alternatively, the individual isomers can be prepared in chiral form or separated chemically from a mixture by forming salts with a chiral acid, such as the individual enantiomers of 10-camphorsulfonic acid, camphoric acid, alpha-bromocamphoric acid, methoxyacetic acid, tartaric acid, diacetyltartaric acid, malic acid, pyrrolidone-5-carboxylic acid, and the like, and then freeing one or both of the resolved bases, optionally repeating the process, so as to obtain either or both substantially free of the other; i.e., in a form having an optical purity of  greater than 95%.
Prevention or inhibition of production of TNFxcex1 by these compounds can be conveniently assayed using anti-TNFxcex1 antibodies. For example, plates (Nunc Immunoplates, Roskilde, DK) are treated with 5 xcexcg/milliliter of purified rabbit anti-TNFxcex1 antibodies at 4xc2x0 C. for 12 to 14 hours. The plates then are blocked for 2 hours at 25xc2x0 C. with PBS/0.05% Tween containing 5 milligrams/milliliter BSA. After washing, 100 xcexcL of unknowns as well as controls are applied and the plates incubated at 4xc2x0 C. for 12 to 14 hours. The plates are washed and assayed with a conjugate of peroxidase (horseradish) and mouse anti-TNFxcex1 monoclonal antibodies, and the color developed with o-phenylenediamine in phosphatecitrate buffer containing 0.012% hydrogen peroxide and read at 492 nm.
Typical compounds of this invention include:
1-phthalimido-1-(3xe2x80x2,4xe2x80x2-diethoxyphenyl)ethane,
1-(1xe2x80x2-oxoisoindolinyl)-1-(3xe2x80x2,4xe2x80x2-diethoxyphenyl)ethane,
1-phthalimido-1-(3xe2x80x2,4xe2x80x2-diethoxyphenyl)propane,
1-(1xe2x80x2-oxoisoindolinyl)-1-(3xe2x80x2,4xe2x80x2-diethoxyphenyl)propane,
1-phthalimido-1-(3xe2x80x2,4xe2x80x2-diethoxyphenyl)butane,
1-(1xe2x80x2-oxoisoindolinyl)-1-(3xe2x80x2,4xe2x80x2-diethoxyphenyl)butane,
1-phthalimido-1-(3xe2x80x2,4xe2x80x2-diethoxyphenyl)-2-phenylethane,
1-(1xe2x80x2-oxoisoindolinyl)-1-(3xe2x80x2,4xe2x80x2-diethoxyphenyl)-2-phenylethane,
1-phthalimido-1-(3xe2x80x2,4xe2x80x2-diethoxyphenyl)-3-pyridylpropane,
1-(1-oxoisoindolinyl)-1-(3,4-diethoxyphenyl)-3-pyridylpropane,
1-phthalimido-1-(3xe2x80x2,4xe2x80x2-diethoxyphenyl)-3-phenylpropane,
1-(1xe2x80x2-oxoisoindolinyl)-1-(3xe2x80x2,4xe2x80x2-diethoxyphenyl)-3-phenylpropane,
1-phthalimido-1-(3xe2x80x2,4xe2x80x2-diethoxyphenyl)-2-pyridylethane,
1-(1xe2x80x2-oxoisoindolinyl)-1-(3xe2x80x2,4xe2x80x2-diethoxyphenyl)-2-pyridylethane,
1-phthalimido-1-(3xe2x80x2,4xe2x80x2-diethoxyphenyl)butane,
1-(1xe2x80x2-oxoisoindolinyl)-1-(3xe2x80x2,4xe2x80x2-diethoxyphenyl)butane,
1-phthalimido-1-(3xe2x80x2,4xe2x80x2-diethoxyphenyl)-2-imidazolylethane,
1-(1xe2x80x2-oxoisoindolinyl)-1-(3xe2x80x2,4xe2x80x2-diethoxyphenyl)-2-imidazolylethane,
1-phthalimido-1-(3xe2x80x2,4xe2x80x2-diethoxyphenyl)-3-methylbutane,
1-(1xe2x80x2-oxoisoindolinyl)-1-(3xe2x80x2,4xe2x80x2-diethoxyphenyl)-3-methylbutane.
The following examples will serve to further typify the nature of this invention but should not be construed as a limitation in the scope thereof, which scope is defined solely by the appended claims.